The long term objective of this project is to understand the molecular mechanisms that regulate gene expression in nitrogen assimilation in eukaryotic organisms. These include the roles of regulatory proteins, compartmentation of enzymes and substrates, and metabolite flux in the overall function of a metabolic pathway. Proline utilization in the yeast Saccharomyces cerevisiae serves as the model system for this study. The proline utilization enzymes are encoded by nuclear genes, translated in the cytosol, and targeted to the mitochondrion where proline is converted to glutamate. Proline, oxygen and a functional electron transport chain are essential for this process. Expression of the structural genes is regulated by an activator called PUT3. This protein is constitutively bound to the promoters of these genes, but activates their transcription only when preferred nitrogen sources are absent and proline is present. The focus of this study is to determine how the signal that proline is available for utilization is transmitted through the PUT3 protein to the transcriptional machinery. The functional domains of the activator protein will be defined by an analysis of mutant PUT3 genes that encode activator-defective proteins, construction of site-directed mutations in specific domains of the gene, exchange of domains with those of GAL4, a well-characterized activator, and measurement of the effects of these alterations on DNA binding, transcriptional activation, proline-responsiveness and dimerization of the protein. The PUT3 protein will be purified using DNA-affinity chromatography and examined for post-translational modifications (phosphorylation, glycosylation, proline-binding and association with other proteins) using standard biochemical methods. A new class of mutations in negative regulators controlling this pathway has been identified and will be characterized by genetic and molecular methods. One member of this class, the product of the PUT6 gene, apparently works through PUT3 in controlling proline utilization, but is not specific to this pathway. Genes that interact with PUT3 will be identified by the isolation of high copy and extragenic suppressors of put3 and PUT3c mutations. This system provides a useful model to understand how changes in the cellular environment are signalled to the elements of a metabolic pathway in eukaryotic organisms.